| [Objective]Low back pain is a common disease with high recurrence rates in adults.Lumbar spine stability decreases after multifidus injury,which is an important factor in the occurrence of low back pain.Prior experiments showed that local injection of bupivacaine caused multifidus injury and noxious stimulus signals activated microglia.Our preliminary hypothesis is that post-injury IL6 and TNF-α may release cytotoxic NO by activating M1 microglia,which may lead to oxidative stress injury in the spinal cord and hippocampus.Electroacupuncture of Weizhong(B1-40)may inhibit excessive microglia activation in the spinal cord and hippocampus by regulating IL-6,TNF-α,CaM,CaMKⅡ and iNOS,improve their antioxidant capacity,and promote multifidus recovery to relieve low back pain.This study explored the mechanism of electroacupuncture on "Weizhong" on oxidative stress injury of the spinal cord and hippocampus mediated by microglia activation through animal and cell experiments,in order to provide some experimental proves for clinical acupuncture treatment of low back pain.[Methods]Animal experiment:SD male rats were randomly divided into blank group,model 1D group,model 3D group,electroacupuncture 1D group and electroacupuncture 3D group.A rat model of lumbar multifidus injury was established by local injection of bupivacaine.In the electroacupuncture group,bilateral "Weizhong" electroacupuncture was used.CaM,CaMKII and iNOS content in the multifidus,spinal cord and hippocampus was detected by ELISA;MDA concentration and SOD activity in spinal cord and hippocampus were detected by biochemical methods;and the expression of CaM,CaMKII and iNOS in hippocampus were detected by Western blot.Cell experiment:Hippocampal microglia were cultured with serum obtained in vivo,and were randomly divided into 20%normal serum group,20%fetal bovine serum group,20%model serum group(model 1D group and model 3D group),20%electroacupuncture serum group(EA 1D group and EA 3D group).The model and electroacupuncture serum groups were further divided into CaM inhibitor group and non-CaM inhibitor group.Microglia M1 was identified by immunohistochemical method;expression of CaM,CaMKⅡ and iNOS were detected by Western blot;expression of CaM mRNA,CaMKⅡ mRNA and iNOS mRNA were detected by RT-PCR;MDA concentration and SOD activity were detected by biochemical method.[Results]Animal experiment results(1)Comparison of IL-6 and TNF-α content in multifidus of rats among all groupsCompared with the blank group,IL-6 and TNF-α content in the model groups 1D and 3D were increased significantly(P<0.01).Compared with the model group 1D,IL-6 and TNF-α content in the EA 1D group were significantly lower(P<0.01),while the TNF-α content in model group 3D was significantly lower(P<0.05).Compared with model group 3D,IL-6 content in EA 3D group was decreased,but there was no significant difference(P>0.05),and the TNF-α content in EA 3D group was significantly lower than that in model 3D group(P<0.01).Compared with the EA 1D group,IL-6 and TNF-α content in the EA 3D group decreased,but there were no significant differences(P>0.05).(2)Comparison of CaM,CaMKⅡ and iNOS content in multifidus,spinal cord and hippocampus of rats among all groupsCompared with the blank group,the content of CaM,CaMKⅡ and iNOS in the hippocampus,spinal cord,and multifidus in the model 1D group and the model group 3D was increased significantly(P<0.01).Compared with the model group 1D,CaM,CaMKⅡ and iNOS content in the hippocampus,spinal cord,and multifidus in the EA 1D group were decreased(P<0.05).Compared with the model group 3D,the content of CaM,CaMKⅡ and iNOS in the hippocampus,spinal cord and multifidus in the EA 3D group was decreased(P<0.05).Compared with the EA ID group,there were no significant differences in CaM content in the multifidus and iNOS content in the hippocampus and spinal cord in the EA 3D group(P>0.05),but CaM and CaMKⅡ content in the hippocampus and spinal cord of the EA 3D group were significantly decreased(P<0.01).(3)Comparison of CaM,CaMKⅡ and iNOS expression in hippocampus of rats among all groupsCompared with the blank group,expression of CaM,CaMKⅡ and iNOS in hippocampus of model 1D group and model 3D group was increased significantly(P<0.01).Compared with model 1D group,the expression of CaM,CaMKⅡ and iNOS in hippocampus of EA 1D group was decreased significantly(P<0.01).Compared with model 3D group,the expression of CaM in hippocampus of EA 3D group was significantly decreased(P<0.01),and the expression of CaMKⅡ and iNOS in hippocampus of EA 3D group was significantly decreased(P<0.05).The expression of CaM,CaMKⅡ and iNOS in hippocampus of EA 3D group were lower than EA 1D group(P<0.05).(4)Comparison of MD A concentration and SOD activity in spinal cord and hippocampus of rats among all groupsCompared with the blank group,MDA concentration in spinal cord and hippocampus increased significantly and SOD activity decreased significantly in model 1D group and model 3D group(P<0.01).Compared with the model 1D group,MDA concentration in the spinal cord was decreased,while SOD activity increased(P<0.05).MDA concentration in the hippocampus decreased significantly and SOD activity increased significantly(P<0.01)in EA 1D group.Compared with the model 3D group,there were no significant differences in MDA concentration in spinal cord and SOD activity in spinal cord and hippocampus(P>0.05)in EA 3D group.However,MDA concentration was decreased significantly in hippocampus in EA 3D group(P<0.01).There was no significant difference in MDA concentration and SOD activity in spinal cord and hippocampus between EA 1D group and EA 3D group(P>0.05).Cell experiment results(1)Identification of M1 type after microglia activationAfter combined induction of LSP and IFN-γ,the typical activation of microglia M1 type and the average optical density of iNOS and CD40 were significantly higher than those in the control group(P<0.01).(2)Comparison of CaM,CaMKⅡ and iNOS expression in microglia among all groups1D interventionCompared with the normal serum group,the expression of CaM in the model serum group increased(P<0.05),and CaMKⅡ expression increased significantly(P<0.01).There was no significant difference in iNOS expression in the model serum group(P>0.05).Compared with the model serum group,there were no significant differences in CaM,CaMKⅡ and iNOS expression between the model serum plus inhibitor group and the electroacupuncture "Weizhong" serum group(P>0.05),while CaM and CaMKⅡ expression in the electroacupuncture "Weizhong" serum plus inhibitor group decreased(P<0.05).Compared with model serum plus inhibitor group,there was no significant difference in CaM,CaMKⅡ and iNOS expression between electroacupuncture "Weizhong" serum group and electroacupuncture "Weizhong" serum plus inhibitor group(P>0.05).3D interventionCompared with the normal serum group,CaM and CaMKⅡ expression in the model serum group was significantly higher(P<0.01).There was no significant difference in iNOS expression in the model serum group(P>0.05).Compared with model serum group,CaM and CaMKⅡ expression in model serum plus inhibitor group,electroacupuncture "Weizhong" serum group and electroacupuncture"Weizhong" serum plus inhibitor group decreased(P<0.05),while iNOS expression showed no significant difference(P>0.05).Compared with model serum plus inhibitor group,there was no significant difference in CaM,CaMKⅡ and iNOS expression in serum group of EA "Weizhong"(P>0.05).(3)Comparison of CaM,CaMKⅡ and iNOS mRNA expression in microglia among all groups1D interventionCompared with the normal serum group,CaM,CaMKⅡ and iNOS mRNA expression in the model serum group was increased significantly(P<0.01).Compared with model serum group,CaM,CaMKⅡ,and iNOS mRNA expression in model serum plus inhibitor group,electroacupuncture "Weizhong" serum group,and electroacupuncture "Weizhong" serum plus inhibitor group was decreased significantly(P<0.01).Compared with model serum plus inhibitor group,there were no significant differences in CaM and CaMKⅡ mRNA expression in electroacupuncture "Weizhong" serum group and electroacupuncture "Weizhong" serum plus inhibitor group(P>0.05),while iNOS mRNA expression was significantly decreased(P<0.01).Compared with the electroacupuncture "Weizhong" serum group,there were no significant differences in CaM mRNA and iNOS mRNA expression in the electroacupuncture "Weizhong"serum plus inhibitors group(P>0.05),while CaMKⅡ mRNA expression was significantly decreased in the electroacupuncture "Weizhong" serum plus inhibitors group(P<0.01).3D interventionCompared with the normal serum group,of CaM,CaMKⅡ and iNOS mRNA expression in the model serum group was increased significantly(P<0.01).Compared with model serum group,CaM mRNA and CaMKⅡ mRNA expression in model serum plus inhibitor group,electroacupuncture" Weizhong" serum group,and electroacupuncture "Weizhong" serum plus inhibitor group were significantly lower(P<0.01),while there were no significant differences in iNOS mRNA expression(P>0.05).C ompared with model serum plus inhibitor group,CaM mRNA and CaMKⅡ mRNA expression in electroacupuncture "Weizhong" serum group and electroacupuncture "Weizhong"serum plus inhibitor group were significantly decreased(P<0.01),while there were no significant differences in iNOS mRNA expression(P>0.05).Compared with the electroacupuncture "Weizhong" serum group,the expression of CaM mRNA in the electroacupuncture "Weizhong" serum plus inhibitor group was decreased(P<0.05),while there were no significant differences in the expression of CaMKⅡ and iNOS mRNA(P>0.05).(4)Comparison of MDA concentration and SOD activity of microglia among all groups1D interventionCompared with normal serum group,MDA concentration and SOD activity in model serum group were increased significantly(P<0.01).Compared with model serum group,MDA concentration in model serum plus inhibitor group and electroacupuncture "Weizhong" serum group were decreased significantly(P<0.01),while there was no significant difference in SOD activity(P>0.05).SOD activity in electroacupuncture "Weizhong" serum plus inhibitor group was increased significantly(P<0.01).Compared with the model serum plus inhibitor group,MDA concentration in the electroacupuncture "Weizhong" serum group and the electroacupuncture "Weizhong" serum plus inhibitor group were decreased significantly(P<0.01).For the electroacupuncture"Weizhong" serum group,SOD activity was not significantly different(P>0.05).The SOD activity in the electroacupuncture "Weizhong" serum plus inhibitor group was increased significantly(P<0.01).Compared with the electroacupuncture "Weizhong" serum group,MDA concentration in the electroacupuncture"Weizhong" serum plus inhibitors group was significantly lower(P<0.01),while there was no significant difference in SOD activity(P>0.05).3D interventionCompared with normal serum group,MDA concentration was significantly increased,while the SOD activity was decreased significantly in model serum group(P<0.01).Compared with model serum group,there were no significant differences in MDA concentration and SOD activity in the model serum plus inhibitor group and electroacupuncture"Weizhong" serum group(P>0.05).The SOD activity in electroacupuncture "Weizhong" serum plus inhibitor group was increased significantly(P<0.01).Compared with model serum plus inhibitor group,there were no significant differences in MDA concentration and SOD activity of electroacupuncture "Weizhong" serum group and MDA concentration of electroacupuncture "Weizhong" serum plus inhibitor group(P>0.05),while the SOD activity of electroacupuncture "Weizhong" serum plus inhibitor group was increased significantly(P<0.01).[Conclusions]In vivo experimentsElectroacupuncture "Weizhong" may relieve lumbar multifidus injury in the rat model by reducing IL-6 and TNF-α content,reducing the content and protein expression of CaM,CaMKⅡ,iNOS in multifidus,spinal cord and hippocampus,and improving the antioxidant capacity of spinal cord and hippocampus.This effect was superior to the spinal cord and hippocampus after 3D intervention,while it was better to the multifidus after 1D intervention.In cell experiments(1)Typical activation of microglia M1 type was induced by LPS and IFN-γ.(2)Electroacupuncture "Weizhong" serum can inhibit the expression of CaM,iNOS protein and gene in microglia,and improve the antioxidative ability in hippocampal microglia.This effect is significant after 3D intervention;When blocked CaM-CaMKⅡ-iNOS signaling pathway with CaM inhibitor,electroacupuncture serum may affect the activation and the antioxidant capacity of hippocampal microglia through other signaling pathways. |
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